Thrombotic thrombocytopenic purpura (TTP) is a disease in which the classical symptoms of thrombocytopenia and microangiopathic, hemolytic anemia, neurological symptoms, disturbances in kidney function, and fever, are observed. Unusually large multimers of the von Willebrand factor (VWF) are found in plasma from TTP patients and are regarded as being the reason for the formation of VWF-rich and platelet-rich thrombi. Endothelial cells release von Willebrand factor in the form of large multimers which, in normal plasma, are cleaved by the combined action of a reductase and a metalloprotease.
In addition, it is already known that patients suffering from congenital or acquired TTP are observed to lack a specific metalloprotease which cleaves VWF between the amino acids Tyr842 and Met843. This metalloprotease has recently been identified as a new member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family and designated ADAMTS-13 Fujikawa, K., Suzuki, H., McMullen, B., Chung, D. (2001) Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood 98: 1662-1666 (hereinafter “1”); Gerritsen; H. E., Robles, R., Lämmele, B., Furlan, M. (2001) Partial amino acid sequence of purified von Willebrand factor-cleaving protease Blood 98 : 1654-1661 (hereinafter “2”); Levy, G. G., Nichols, W. C., Lian, E. C. Foroud, T., McClintick, J. N., McGee, B. M., Yang, A. Y., Siemieniak, DR., Stark. K. R., Gruppo, R., Sarode, R., Shurin, S. B., Chandrasekaran. V., Stabler, S. P., Sabio. H., Bouhassira, E. E., Upshaw, J. D., Ginsburg, D., Tsai, H. M. (2001) Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413: 488-494 (hereinafter “3”).
In that which follows, the VWF-cleaving protease activity of ADAMTS-13 is simply termed ADAMTS-13 activity. ADAMTS-13 activity is normally measured by incubating a VWF sample, which has been treated with urea or guanidium hydrochloride, with dilute plasma at low ionic strength. The proteolysis is detected by means of a multimer analysis using SDS agarose gel electrophoresis or by means of fragment analysis using SDS polyacrylamide gel electrophoresis and subsequent immunoblotting, that is detecting the proteins on a cellulose membrane by means of an antigen-antibody reaction Furlan, M., Robles, R., Lämmle, B., (1996) Partial purification and characterisation of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 10: 4223-4234 (hereinafter “4”): Tsai, H. M. (1996) Physiologic Cleavage of von Willebrand factor by a Plasma Protease is dependent on its confirmation and requires Calcium ion. Blood 10: 4235-4244 (hereinafter “5”). The ADAMTS-13-catalyzed degradation of the von Willebrand factor can also be determined by measuring the collagen-binding activity of the VWF (WO-A 00/50904) or by carrying out a specific, bilateral ELISA detection Obert B, Tout H. Veyradier A. Fressinaud F, Meyer D, Girma JP (1999) Estimation of the Willebrand factor-cleaving protease in plasma using monoclonal antibodies to VWF. Thromb Haemost 82: 1382-1385 (hereinafter“6”). A recombinant monomeric VWF, which has been labeled at the N terminus with a green fluorescent protein, has also recently been described for the purpose of determining the proteolysis Raife TJ, Atkinsons B, Christopherson P. Jozwiak M, Montgomery RR (2001) Recombinant, truncated monomeric von Willebrand factor (VWF) for the study of VWF proteolysis. Thromb Haemost, Suppl July: Abstract#1667 (hereinafter “7”).
The conventional electrophoretic methods can only be carried out in specialized research laboratories since implementing the tests requires special laboratory equipment and the requisite expertise. While the collagen-binding test (WO-A 00/50904) and the specific ELISA (6) for detecting the proteolytic activity of ADAMTS-13 simplify the determination of ADAMTS-13 activity, they can likewise only be carried out in the laboratories which have at their disposal the appropriate equipment and the necessary knowhow. Furthermore, the bilateral ELISA requires specific monoclonal antibodies which are only available in a few laboratories since they cannot be obtained commercially. The object therefore presented itself of developing a simple method for determining ADAMTS-13 activity. This novel method was to make it possible to quantify ADAMTS-13 activity in blood plasma and other body fluids (e.g. blood serum, and saliva), and other media, in a reliable and timely manner. It was to be utilizable in any routine clinical coagulation laboratory and therefore not require any special laboratory equipment, special technical knowhow or reagents which were not available commercially. Furthermore, the novel method was to permit an automation in automatic coagulation machines which was as far reaching as possible in order to make it possible to achieve a high sample throughput at low operational cost. Since it has by now been demonstrated that low ADAMTS-13 activities can also be observed in diseases other than TTP, such a method was to make it possible to differentiate between the severe ADAMTS-13 deficiency which is characteristic of TTP and mild ADAMTS-13 deficiency. Kasper. C. K. (1991) Laboratory tests for factor VIII inhibitors, their variation, significance and interpretation. Blood Coagul Fibrinolysis 2: 7-10 (hereinafter “8”) An early diagnosis, and consequently a rapid initiation of plasmapheresis therapy, essentially determines the clinical course of what is frequently a life-threatening TTP episode. For this reason, the novel method was to enable ADAMTS-13 activity to be determined rapidly and as comprehensively as possible. Timely determination of the ADAMTS-13 activity is also essential because of the existence of alternative therapy options. In particular, the method was to enable an inhibitor against ADAMTS-13 to be detected rapidly, or to enable the inhibitor titer to be determined, since different treatment possibilities (e.g. rituximab or immunoadsorption) ensue from this. The method was also, in particular, to make it possible to differentiate between congenital and acquired TTP. In addition to this, timely ADAMTS-13 activity determination, which is capable of being carried out routinely, is the prerequisite for using the recombinant ADAMTS-13 which is potentially available (W0242441). The method was not only to permit timely diagnosis, and monitoring of the therapy, of TTP patients but, in addition, reliable quantification of the ADAMTS-13 activity in any arbitrary media. Since ADAMTS-13 is an important regulator of VWF, and consequently a significant factor in hemostasis, the novel method was to be applicable in diverse studies of the importance of ADAMTS-13-catalyzed proteolysis of VWF in healthy subjects and patients suffering from different diseases.